HIV-1 and other primate lentiviruses encode accessory genes that serve to enhance virus replication and counteract host antiviral factors. These restriction factors have the capacity to limit virus replication and influence cross-species transmissions of lentiviruses. Moreover, the interactions between viral accessory proteins and host restriction factors are a potential source of novel drug interventions for HIV therapy. Vpx and Vpr are two related lentiviral accessory proteins. While all primate lentiviruses encode Vpr, only two of them encode Vpx as well. Vpx is critical for the ability of primate lentiviruses to efficiently infect monocytes, dendritic cells, macrophages, and resting T cells, while Vpr has many ascribed functions including the ability to arrest cells in the G2 phase of the cell cycle. Recently, the target of Vpx has been identified as the host restriction factor SAMHD1 which is targeted for degradation by Vpx. An evolutionary analysis to understand the functional relationship of Vpx and Vpr showed that Vpx and Vpr have overlapping functions. Vpx proteins from diverse lentiviruses as well as Vpr proteins from some lentiviruses both have the ability to target their host species' SAMHD1 for degradation. However, HIV-1 Vpr does not interact with SAMHD1, and therefore the importance of this virus-host interaction remains a mystery. In this grant we will determine the importance of the SAMHD1 interaction by examining the host-virus evolution in a natural infection study of African green monkeys and their lentirviral infections. Furthermore, we will determine how Vpx/Vpr and SAMHD1 have co-evolved to recognize and escape each another by determining the molecular basis by which SAMHD1 proteins from different primate hosts are recognized by different Vpx/Vpr proteins. We will also examine the interactions of Vpx from a human pathogen, HIV-2 with human SAMHD1 and will explore the possibility that some HIV-1 strains also encode a Vpr protein that is capable of degrading SAMHD1. Finally, we will test the hypothesis that new functions for Vpr and Vpx have evolved on top off pre-existing functions by determining the ancestral function of Vpr and by determining how some Vpr proteins can have multiple functions.